1. Field of the Invention
This invention relates to the field of therapeutic peptides for the prevention and treatment of disorders or diseases resulting from abnormal formation of amyloid or amyloid-like deposits, such as, but not limited to, prion-related encephalophathies, Alzheimer""s dementia or disease (AD), and other amyloidosis disorders. This invention also relates to the use of the peptides in preventing the formation of or in promoting the redissolution of these insoluble amyloid or amyloid-like deposits.
2. Description of the Background Art
Alzheimer""s disease (AD) is the most common form of dementia in adults (C. Soto et al. J. Neurochem. 63:1191-1198, 1994), constituting the fourth leading cause of death in the United States. Approximately 10% of the population over 65 years old is affected by this progressive degenerative disorder that is characterized by memory loss, confusion and a variety of cognitive disabilities. One of the key events in AD is the deposition of amyloid as insoluble fibrous masses (amyloidogenesis) resulting in extracellular neuritic plaques and deposits around the walls of cerebral blood vessels. The main component of amyloid is a 4.1-4.3 kDa hydrophobic peptide, named amyloid xcex2-peptide (Axcex2), that is codified in chromosome 21 as part of a much longer amyloid precursor protein APP (Muller-Hill and Beyreuther, Ann. Rev. Biochem. 38:287-307, 1989). The APP starts with a leader sequence (signal peptide), followed by a cysteine-rich region, an acidic-rich domain, a protease inhibitor motif, a putative N-glycosylated region, a transmembrane domain, and finally a small cytoplasmic region. The Axcex2 sequence begins close to the membrane on the extracellular side and ends within the membrane. Two-thirds of Axcex2 faces the extracellular space, and the other third is embedded in the membrane (Kang et al. Nature 325:503-507, 1987; Dyrks et al. EMBO J. 7:949-957, 1988). Several lines of evidence suggest that amyloid may play a central role in the early pathogenesis of AD.
Evidence that amyloid may play an important role in the early pathogenesis of AD comes primarily from studies of individuals affected by the familial form of AD (FAD) or by Down""s syndrome. Down""s syndrome patients have three copies of APP gene and develop AD neuropathology at an early age (Wisniewski et al., Ann. Neurol. 17:278-282, 1985). Genetic analysis of families with hereditary AD revealed mutations in chromosome 21, near or within the Axcex2 sequence (Forsell et al., Neurosci. Lett. 184:90-93, 1995). Moreover, recently it was reported that transgenic mice expressing high levels of human mutant APP progressively develop amyloidosis in brain (Games et al., Nature 373:523-527, 1995). These findings appear to implicate amyloidogenesis in the pathophysiology of AD.
Recently, the same peptide that forms amyloid deposits in AD brain was also found in a soluble form (sAxcex2) normally circulating in the human body fluids (Seubert et al., Nature 359:355-327, 1992; Shoji et al., Science 258:126-129, 1992). Zlokovic et al., Biochem. Biophys. Res. Commun. 205:1431-1437 (1994), reported that the blood-brain barrier (BBB) has the capability to control cerebrovascular sequestration and transport of circulating sAxcex2, and that the transport of the sAxcex2 across the BBB was significantly increased when sAxcex2 was perfused in guinea pigs as a complex with apolipoprotein J (apoJ). The sAxcex2-apoJ was found in normal cerebrospinal fluid (CSF; Ghiso et al. Biochem. J. 293:27-30, 1994) and in vivo studies indicated that sAxcex2 is transported with apoJ as a component of the high density lipoproteins (HDL) in normal human plasma (Koudinov et al., Biochem. Biophys. Res. Commun. 205:1164-1171, 1994). It was also recently reported by Zlokovic et al., Proc. Natl. Acad. Sci. USA 93:4229-04233 (1996), that the transport of sAxcex2 across the BBB was almost abolished when the apoJ receptor gp330 was blocked. It is believed that the conversion of sAxcex2 to insoluble fibrils is initiated by a conformational or proteolytic modification of the 2-3 amino acid longer soluble form. It has been suggested that the amyloid formation is a nucleation-dependent phenomena in which the initial insoluble xe2x80x9cseedxe2x80x9d allows the selective deposition of amyloid (Jarrett et al., Biochem. 32 :4693-4697, 1993).
Peptides containing the sequence 1-40 or 1-42 of Axcex2 and shorter derivatives can form amyloid-like fibrils in the absence of other protein (Soto et al., J. Neurochem. 63:1191-1198, 1994), suggesting that the potential to form amyloid resides mainly in the structure of Axcex2. The relation between the primary structure of Axcex2 and its ability to form amyloid-like fibrils was analyzed by altering the sequence of the peptide. Substitution of hydrophilic residues for hydrophobic ones in the internal Axcex2 hydrophobic regions (amino acids 17-21) impaired fibril formation (Hilbich et al., J. Mol. Biol. 228:460-473, 1992), suggesting that Axcex2 assembly is partially driven by hydrophobic interactions. Indeed, larger Axcex2 peptides (Axcex21-42/43) comprising two or three additional hydrophobic C-terminal residues are more amyloidogenic (Jarrett et al., Biochem 32:4693-4697, 1993). Secondly, the conformation adopted by Axcex2 peptides is crucial in amyloid formation. Axcex2 incubated at different pH, concentrations and solvents has mainly an a-helical (random coil) or a xcex2-sheet secondary structure (Barrow et al., J. Mol. Biol. 225:1075-1093, 1992: Burdick""et al., J. Biol. Chem. 267:546-554, 1992; Zagorski et al., Biochem. 31:5621-5631, 1992). The Axcex2 peptide with xcex1-helical or random coil structure aggregates slowly; Axcex2 with xcex2-sheet conformation aggregates rapidly (Zagorski et al., Biochem. 31:5621-5631, 1992; Soto et al., J. Biol. Chem. 270:3063-3067, 1995; Soto and Castano, Biochem. J. 314:701-707, 1996). The importance of hydrophobicity and xcex2-sheet secondary structure on amyloid formation also is suggested by comparison of the sequence of other amyloidogenic proteins.
Analysis of Axcex2 aggregation by turbidity measurements indicates that the length of the C-terminal domain of Axcex2 influences the rate of Axcex2 assembly by accelerating nucleus formation (Jarrett et al., Cell 73:1055-1058, 1993 ). Thus, the C-terminal domain of Axcex2 may regulate fibrillogenesis. However, in vitro modulators of Axcex2 amyloid formation, such as metal cations (Zn, Al) (Bush et al., Science 265:1464-1467, 1994; Exley et al., FEBS Lett. 324:293-295, 1993) heparin sulfate proteoglycans, and apoliprotein E (Strittmatter et al., Proc. Natl. Acad. Sci. USA 90:1977-1981, 1993) interact with the 12-28 region of Axcex2. Moreover, mutations in the xcex2 PP gene within the N-terminal Axcex2 domain yield analogs more fibrillogenic (Soto et al., 1995, supra; Wisniewski et al., Biochem. Biophys. Res. Commun. 179:1247-1254, 1991). Finally, while the C-terminal domain of Axcex2 invariably adopts a xcex2-strand structure in aqueous solutions, environmental parameters determine the existence of alternative conformation in the Axcex2 N-terminal domain (Barrow et al., 1992, supra; Soto et al., 1995, supra; Burdick et al., 1992, supra). Therefore, the N-terminus may be a potential target site for inhibition of the initial random coil to xcex2-sheet conformational change.
The emerging picture from studies with synthetic peptides is that Axcex2 amyloid formation is dependent on hydrophobic interactions of Axcex2 peptides adopting an antiparallel xcex2-sheet conformation and that both the N- and C-terminal domains are important for amyloid formation. The basic unit of fibril formation appears to be the conformer adopting an antiparallel xcex2-sheet composed of strands involving the regions 10-24 and 29-40/42 of the peptide (Soto et al., 1994, supra). Amyloid formation proceeds by intermolecular interactions between the xcex2-strands of several monomers to form an oligomeric xcex2-sheet structure precursor of the fibrillar xcex2-cross conformation. Wood et al., Biochemistry 34:724-730 (1995), reported the insertion of aggregation-blocking prolines into amyliod proteins and peptides, as exemplified by test peptides having the amino acid sequences of SEQ ID NOs:50-65, to prevent aggregation of such proteins and peptides. In this manner, the authors suggest that novel proteins can be designed to avoid the problem of aggregation as a barrier to their production without affecting the structure or function of the native protein. Thus, Wood et al. seek to produce novel proteins that would not aggregate during recombinant protein production and purification by inserting aggregation-blocking prolines into these novel prolines. The inhibitory peptides of the present invention, which inhibit the formation of amyloid deposits from native Axcex2 as opposed to solely preventing aggregation of a proline-modified peptide or protein, are not intended to include the peptides of Wood et al. having the amino acid sequences SEQ ID NOs:50-65.
To date there is no cure or treatment for AD and even the unequivocal diagnosis of AD can only be made after postmortem examination of brain tissues for the hallmark neurofibrillary tangles (NFT) and neuritic plaques. However, there are several recent publications outlining strategies for the treatment of Alzheimer""s disease.
Heparin sulfate (glycosoaminoglycan) or the heparin sulfate proteoglycan, perlecan, has been identified as a component of all amyloids and has also been implicated in the earliest stages of inflammation-associated amyloid induction. Kisilevsky et al., Nature Medicine 1(2):143-148, (1995) describes the use of low molecular weight (135-1,000 Da) anionic sulfonate or sulfate compounds that interfere with the interaction of heparin sulfate with the inflammation-associated amyloid precursor and the xcex2-peptide of AD. Heparin sulfate specifically influences the soluble amyloid precursor (SAA2) to adopt an increased xcex2-sheet structure characteristic of the protein-folding pattern of amyloids. These anionic sulfonate or sulfate compounds were shown to inhibit heparin-accelerated Alzheimer""s Axcex2 fibril formation and were able to disassemble preformed fibrils in vitro as monitored by electron micrography. Moreover, when administered orally at relatively high concentrations (20 or 50 mM), these compounds substantially arrested murine splenic inflammation-associated amyloid progression in vivo in acute and chronic models. However, the most potent compound, poly- (vinylsulfonate), was acutely toxic.
Anthracycline 4xe2x80x2-iodo-4xe2x80x2-deoxy-doxorubicin (IDOX) has been observed clinically to induce amyloid resorption in patients with immunoglobin light chain amyloidosis (AL) Merlini et al., Proc. Natl. Acad. Sci. USA 92:2959-2963 (1995), elucidated its mechanism of action. IDOX was found to bind strongly via hydrophobic interactions to two distinct binding sites (Scatchard analysis) in five different tested amyloid fibrils, inhibiting fibrillogenesis and the subsequent formation of amyloid deposits in vitro. Preincubation of IDOX with amyloid enhancing factor (AEF) also reduced the formation of amyloid deposits. Specific targeting of IDOX to amyloid deposits in vivo was confirmed in an acute murine model. This binding is distinct from heparin sulfate binding as removal of the glycosaminoglycans from extracted amyloid fibrils with heparinases did not modify IDOX binding. The common structural feature of all amyloids is a xcex2-pleated sheet conformation. However, IDOX does not bind native amyloid precursor light chains which suggests that the xcex2-pleated sheet backbone alone is not sufficient to form the optimal structure for IDOX binding, and that it is the fibril cross-xcex2-sheet quaternary structure that is required for maximal IDOX binding. It has been found that the amount of IDOX extracted from spleens is correlated with amyloid load and not circulating serum precursor amyloid levels. IDOX, however, is also extremely toxic.
The regulation and processing of amyloid precursor protein (APP) via inhibition or modulation of phosphorylation of APP control proteins has also been investigated in U.S. Pat. No. 5,385,915 and WO 9427603. Modulating proteolytic processing of APP to nucleating forms of AD has also been examined in AU 9338358 and EP569777. WO 95046477 discloses synthetic peptides of composition X-X-N-X (SEQ ID NO:69) coupled to a carrier, where X is a cationic amino acid and N is a neutral amino acid, which inhibit Axcex2 binding to glycosoaminoglycan. Peptides containing Alzheimer""s Axcex2 sequences that inhibit the coupling of xcex1-1-antichymotrypsin and Axcex2 are disclosed in WO 9203474.
Prions are the infectious particles responsible for a group of fatal neurodegenerative diseases known as spongiform encephalopathies (for reviews, see Prusiner and DeArmond, Annu. Rev. Neurosci. 17:311-339, 1994; Prusiner, Science 252:1515-1522, 1991). Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler syndrome (GSS) and fatal familial insomnia are all human neurodegenerative diseases caused by prions and are frequently transmissible to laboratory animals. Familial CJD and GSS are also genetic disorders. In addition to the prion diseases in humans, four disorders of animals are included in this type of disease. Scrapie in sheep and goats is the most common of the prion diseases. Bovine spongiform encephalopathy (BSE), also known as the xe2x80x9cmad cow diseasexe2x80x9d, transmissible mink encephalopathy, and chronic wasting disease of captive mule deer and elk are all thought to result from the ingestion of scrapie-infected animal products.
To date there is no cure or effective treatment for prion-related diseases. The infectious agent causing prion-related diseases differ from bacteria, fungi, parasites, viroids and viruses in that no DNA is needed and it apparently only consists of protein. The principal component of prions is the glycoprotein called PrPsc. Prion replication is hypothesized to occur when PrPsc in the infecting inoculum interacts specifically with host PrPc (normal cellular PrP isoform), catalyzing its conversion to the pathogenic form of the protein (Cohen, F. E. et al., Science 264:530-531, 1994). It is postulated that this conversion takes place spontaneously in PrP molecules carrying mutations that have been linked to familial forms of prion disease.
The cellular prion protein (PrPc) is a sialoglycoprotein encoded by a gene that in humans is located on chromosome 20 (Oesch, B. et al., Cell 40:735-746, (1985); Basler, K. et al., 46:417-428 (1986); Liao, Y. J. et al., Science 233:364-367 (1986); Meyer, R. K. et al., Proc. Natl. Acad. Sci. USA 83:2310-2314 (1986); Sparkes, R. S. et al., Proc. Natl. Acad. Sci. USA 83:7358-7362 (1986); Bendheim, P. E. et al. J. Infect. Dis. 158:1198-1208 (1988); Turk, E. et al. Eur. J. Biochem. 176:21-30 (1988)). The PrP gene is expressed in neural and non-neural tissues, the highest concentration of mRNA being in neurons (Chesebro, B. et al., Nature 315:331-333 (1985); Kretzschmar, H. A. et al., Am. J. Pathol. 122:1-5 (1986); Brown, H. R. et al., Acta Neuropathol. 80:1-6 (1990); Cashman, N. R. et al., Cell 61:185-192 (1990); Bendheim, P. E., Neurology 42:149-156 (1992)).
The translation product of the PrP gene consists of 253 amino acids in humans (Kretzschmar, H. A. et al., DNA 5:315-324 (1986); Pucket, C. et al., Am. J. Hum. 49:320-329 (1991)), 254 in hamster and mice or 256 amino acids in sheep and undergoes several post-translational modifications. In hamsters, a signal peptide of 22 amino acids is cleaved at the N-terminus, 23 amino acids are removed from the C-terminus on addition of a glycosyl phosphatidylinositol (GPI) anchor, and asparagine-linked oligosaccharides are attached to residues 181 and 197 in a loop formed by a disulfide bond (Turk, E. et al., Eur. J. Biochem. 176:21-30 (1988); Hope, J. et al., EMBO J. 5:2591-2597 (1986); Stahl, N. et al., Cell 51:229-b 240 (1987); Stahl, N. et al., Biochemistry 29:5405-5412 (1990); Safar, J. et al., Proc. Natl. Acad. Sci. USA 87:6377 (1990)).
In prion-related encephalopathies, PrPc (normal cellular isoform) is converted into an altered form designated PrPSc, that can be experimentally distinguished from PrPc by the following three properties (Cohen et al. Science 264:530-531 (1994): (1) PrPsc is insoluble in physiological solvents and forms aggregates; (2) PrPsc is partially resistant to proteolytic degradation by proteinase K in that only the N-terminal 67 amino acids are removed by proteinase K digestion under conditions in which PrPc is completely degraded, and which results in a N-terminally truncated form known as PrP27-30; and (3) PrPsc has an alteration in protein conformation from xcex1-helical for PrPc to an altered form which is rich in xcex2-sheet secondary structure.
Several lines of evidence indicate that PrPsc is a major and necessary component of the infectious prion (reviewed in Prusiner, S. B. Science 252:1515-1522, 1991) and are as follows: (a) copurification of PrP27-30 and scrapie infectivity were determined by biochemical methods where the concentration of PrP27-30 is proportional to prion titer; (b) kinetics of proteolytic digestion of PrP27-30 and infectivity are similar; (c) copurification of PrPsc and infectivity were observed using immunoaffinity; (d) infectivity was neutralized by anti-PrP antibodies; (e) detection of PrPsc were only detected in clones of cultured cells producing infectivity; (f) most, if not all, of the familial cases of PrP-related disorders are linked to mutations in the PrP gene; (g) mice expressing PrP genes with point mutations linked to GSS spontaneously develop neurologic dysfunction, spongiform brain degeneration and astrocytic gliosis; (h) the species barrier to prion transmission from hamster to mouse could be overcome by introducing a Syriam hamster PrP transgene into the recipient mouse line; and (i) mice devoid of PrP gene are resistant to scrapie infection, developing neither symptoms of scrapie nor allowing propagation of the infectious agent. It has also been established that the protease-resistant core of PrPsc is the major structural protein of amyloid fibrils that accumulate intracerebrally in some of these conditions (Brendheim, P. E. et al., Nature 310:418-421 (1984); DeArmond, S. J. et al., Cell 41:221-235 (1985); Kitamoto, T. et al., Ann. Neurol. 20:204-208 (1986); Robert, G. W. et al., N. Engl. Med. 315:1231-1233 (1986); Ghetti, B. et al., Neurology 39:1453-1461 (1989); Tagliavini, F. et al., EMBO J. 10:513-519 (1991); Kitamoto, T. et al., Neurology 41:306-310 (1991)).
Although there are obvious differences in the etiology and pathogenesis of PrP-related diseases and Alzheimer""s disease (AD), a remarkable number of similarities exist (for reviews, see Kelly, J. W. Curr. Opin. Struct. Biol. 6:11-17, 1996; Castano, E. M. and Frangione, B. Curr. Opin. Neurol. 8:279-285, 1995; Diringer, H. Exp. Clin. Immunogenet. 9:212-229, 1992; DeArmond, S. J. Curr. Opin. Neurol. 6:872-881, 1993), namely: (a) the clinical symptoms of both diseases are similar, including memory loss, behavioral abnormalities, cognitive problems and dementia; (b) both disorders are characterized clinically by age-related sporadic and familial forms of the disease; (c) an abnormal form of a neuronal membrane protein (Amyloid-xcex2 precursor protein and PrP) appears to play a key role in the pathogenesis of both diseases; (d) both Axcex2 and PrP are amyloidogenic and neurotoxic; (e) an important part of the cases affected by familial prion disease typically develop neuritic plaques similar to the AD plaques, but containing PrP (instead of Axcex2) amyloid cores; (f) a hallmark event in both diseases is the conformational transition from an xcex1-helical-random coil structure to a xcex2-sheet conformation in either PrP or Axcex2.
While amyloid deposition is not a general characteristic of PrP-related diseases, extensive evidence suggests that the disorder is caused by a disease-specific posttranslational modification of a normal protein isoform (cellular PrP or PrPc) which results in the abnormal scrapie PrP (PrPsc) (Prusiner, S. B. Science 252:1515-1522, 1991). Chemical differences have not been detected between the two PrP isoforms; they only differ in their conformations. For instance, the major secondary structure of PrPsc is xcex2-ple-ated sheet, as opposed to the predominance of xcex1-helix in PrPc (Pan, K. M. et al. Proc. Natl. Acad. Sci. (USA) 90:10962-10966, 1993). As discussed above, while amyloid or amyloid-like deposits are not observed in all subjects with a PrP-related disease, the pathological xcex2-sheet-rich conformation of PrPsc as an abnormal precursor of amyloid or amyloid-like deposits however are always present.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
The present invention relates to novel inhibitory peptides capable of interacting or binding to a hydrophobic xcex2-sheet forming cluster on a protein or peptide which forms amyloid or amyloid-like deposits so as to inhibit or structurally block the abnormal folding of the protein or peptide into a pathological xcex2-sheet structure to form an amyloid or amyloid-like deposit, or a precursor thereof, such as is observed in Alzheimer""s disease, amyloidosis disorders, prion-related encephalophathies, etc. The peptide includes a hydrophobic portion having one or more xcex2-sheet blocking amino acid residues, and may also include charged amino acids at one or both ends of the peptide. Such inhibitory peptides have a low probability of adopting a xcex2-sheet conformation and are capable of associating with said hydrophobic xcex2-sheet forming cluster on the protein or peptide to structurally block and inhibit the abnormal folding thereof into amyloid or amyloid-like deposits, or pathological xcex2-sheet precursors of amyloid or amyloid-like deposits.
The present invention also relates to a method of preventing or treating a disorder or disease associated with the formation of amyloid or amyloid-like deposits involving the abnormal folding of a protein or peptide having a hydrophobic xcex2-sheet forming cluster into a xcex2-sheet structure, by administering an effective amount of such an inhibitory peptide to a subject in need thereof to prevent or reverse the abnormal folding of the protein or peptide into amyloid or amyloid-like deposits or pathological xcex2-sheet precursors thereof.
The present invention further relates to pharmaceutical compositions for the prevention or therapeutic treatment of disorders or diseases associated with abnormal protein folding into amyloid or amyloid-like deposits and pathological xcex2-sheet-rich precursors thereof, using such inhibitory peptides.
The present invention also relates to a method for detecting disorders or diseases associated with amyloid or amyloid-like fibril deposits and pathological xcex2-sheet-rich precursors thereof.